Comparison of Cochlear Microphonics Magnitude with Broad and Narrow Band Stimuli in Healthy Adult Wistar Rats.

Objective
Cochlear microphonic (CM) is a cochlear AC electric field, recorded within, around, and remote from its sources. Nowadays it can contribute to the differential diagnosis of different auditory pathologies such as auditory neuropathy spectrum disorder (ANSD). This study compared CM waveforms (CMWs) and amplitudes with broad and narrow band stimuli in 25 healthy male young adults Wistar rats.


Materials & Methods
This experimental study was accomplished in the School of Rehabilitation Sciences of Iran University of Medical Sciences, Tehran, Iran (April, 2016). Using an extratympanic technique in ECochG (Electrocochleography) recording, CMWs in response to click and tonal stimuli with different octave frequencies were recorded at a high intensity level in subjects. The CMW amplitudes were calculated by a graphical user interface (GUI) designed in MATLAB.


Results
The CMW magnitude increased upon an increase in bandwidth stimulation. CM amplitude with click stimulation was larger than tonal stimuli. Across tonal stimuli, the CMW amplitudes at lower frequency tones were larger than those at higher frequency tones. Those findings were statistically significant (P<0.001).


Conclusion
CMW amplitude with click as broadband stimulus was larger than those with tone bursts as narrowband stimulation. Click stimulation due to the width of spectral involves greater regions of cochlear partition. Therefore, CMW most likely is a reflection of spatial summation of voltage drops generated by hair cell groups in response to acoustic stimulation. In order to production nature of CM potentials as well as their very small magnitudes especially with tonal stimuli, thus, we recommend using click stimulation for CM potential recording.

cochlear amplification function (16). Despite the advantages of OAE, there are some limitations including restricted measurement and high levels of artifacts due to background noise involving acoustic and physiologic noise (10).
The ABR recording is the most common test for threshold estimation, especially for pediatric and neurotologic purposes for which OAE cannot be used. The other limitation of OAE is vulnerability to middle ear (ME) disease, such as effusion otitis media, which has a high prevalence in infants and children (15).
In contrast to OAE, CM is an electrical signal and is not influenced by acoustic noise. It can be measured simultaneously with ABR recording, therefore saving money and time (10). The measurable frequency range in CM is greater than that of OAE and the former is resistant to ME pathologies. In addition, some studies reported greater stability of CM than of OAE in ANSD; OAE disappeared during the time course of the disorder but CM even showed a high amplitude and long duration in some patients (12,14).

Similar generators have been indicated for both
OAE and CM; however, the cochlear mechanisms underlying these responses are different. For instance, their dual behavior against crossed olivocochlear bundle function can confirm this claim. Stimulation of an efferent auditory system increases CM amplitude whereas the magnitude of OAE decreases (14). Moreover, in a prestin knockout mouse model study, CM was similar to that of the wild-type mice. CM was not influenced by the cochlear amplifier (6). Hence, application of CM and OAE provides more information about the functional nature of the cochlea and gives an extended view of its analysis.

Introduction
The cochlear microphonic (CM) is an alternative current (AC) voltage and one of the auditory receptor potentials (1,2). Its generators are mechanoelectrical transduction currents through the population of hair cells (mainly outer hair cells (OHCs)) and the driving force of endocochlear potential (EP) evoked in response to auditory stimuli. It is elicited by basilar membrane (BM) displacement and stereocilia deflection (1,3,4). On the other hand, CM represents extracellular voltage alterations in OHCs dominated basally and possibly some receptor currents of Inner hair cells (IHCs) (5). CM is a preneural, sustained response and follows the waveform of the acoustic stimuli (6). This product of cochlear hair cells can be recorded in humans and experimental animals at several recording sites (7). Although more than 80 yr have passed since the discovery of CM, its application in clinical settings was restricted due to limitations such as the use of invasive transtympanic electrode array and electromagnetic interaction (8)(9)(10).
Currently, CM recording has attracted new interest because it has an important role in the diagnosis of auditory neuropathy spectrum disorder (ANSD) (11)(12)(13)(14). This disorder is characterized by absent or severely abnormal ABR with OAE and/or CM preservation, which are indicators of OHCs integrity. The incidence of ANSD is higher in infants especially treated in the neonatal intensive unit care (NICU) (12)(13)(14)(15). Three subcutaneous needle electrodes were placed at vertex (non-inverting), under the right (inverting) and the left (ground) ears as in an ABR electrode array (18). If the electrode impedance at three sites was lower than 5 kΩ and the interelectrode impedance was <2 kΩ, then calibrated stimuli were delivered by loudspeaker located 5 cm from the right ear at 80 dB SPL, with the stimuli and acquisition parameters including: 7.1/ sec as the repetition rate, a 5.33-ms time window with 1-ms prestimulation, sampling of 256 points, amplified×100000, a bandpass filter of 100-1500 Hz, and an average of 1000 waveforms. To confirm appropriate recording methods and true CM recordings, the following approaches were used: phase CMWs inversed with alterations in stimulus single polarity involving rarefaction and condensation, alternating polarity eliminated CMWs (7, 15), and they followed the stimulation duration and frequency (15,18). CMWs that met these criteria were accepted as true.
At each stimulus set, two records were obtained and stored for offline analysis.    In addition, Figure 2 presents the CMWs for all tested stimuli in one subject. As was observed, CMWs follow stimulation frequency, polarity, and waveform.

Comparison of Cochlear Microphonics Magnitude with Broad and Narrow Band Stimuli in Healthy Adult Wistar Rats
Five traces from top to bottom represent click, 2000, 4000, 8000, and 16000 Hz waveforms, respectively, in response to stimulus intensity level at 80 dB SPL with rarefaction polarity.

Discussion
The aim of the current study was to compare CM amplitude with different stimuli (Click as broadband stimulus and tone bursts as narrowband stimuli at different octave frequencies). Click has been known as a short time stimulus which spreads over a wide range of frequencies, whereas the time of tonal stimulus is longer, but has a narrow frequency spread (15). Our question was how does CM behave in terms of its amplitude and waveform in response to various stimuli at a constant highintensity level (80 dB SPL) in healthy young adult Wistar rats? Rats are attractive as auditory system models. The reasons for this are their availability and their similar genetic features as well as hearing characteristics to those of humans. Therefore, auditory results in a rat model can be extrapolated to human hearing (1).
The findings of the current study revealed that the CM amplitude with click stimulus was generally larger than that with tonal stimuli. Across four tonal stimuli, there was an inverse relationship between the CM amplitude and frequency. In other words, higher frequencies showed smaller CM amplitude than lower frequencies did, and this difference was statistically significant. Regarding the polarity and waveforms of stimulation, CMWs is not frequency-specific and its traveling wave is spread along the cochlear partition from base to apex and involves more hair cells than narrow band stimuli does. In fact, CM is a reflection of the spatial summation of hair cell receptor currents (4,17). These physiologic properties can infer that greater numbers of hair cell groups contribute to CM production during broadband stimulation and with narrow band lower frequencies rather than with high frequencies, and this leads to larger CM amplitude for click stimulus and lower frequencies.
The other hypothesis explaining these findings is the nature of the intrinsic low-pass filter of hair cells (8); when stimulation frequency increases, AC potential decreases despite the fact that DC potential increases. In addition, in vivo study in mice has shown a variety of stimulus intensity-CM magnitude functions for different frequencies (17).
The saturation level for low frequency was higher than that of the high-frequency tones. That is, the amplitude of CM potential at low frequencies in mice grew with increasing stimulation level. The present study was conducted with a high-intensity level; therefore, low frequencies were able to increase the AC field more than high frequencies were.
Limited previous studies using animal models or human research reported that lower frequencies had larger CM amplitude than higher frequencies did (17,21). However, to our knowledge, a comparison of CM amplitude according to click and tonal stimuli had not yet been conducted.
In conclusion, the CM amplitude was influenced by the bandwidth of the stimulation. CM amplitude with click was larger than tonal stimuli.
In addition, across tonal stimuli with different octave frequencies, there was inverse relationship between CM amplitude and frequency. Since, click stimulus spreads over a wider range of frequencies, and traveling wavelength of low-frequency tonal stimuli are longer than high-frequency ones, this very small AC cochlear potential is a reflection of the spatial summation of hair cell groups according to traveling wave propagation along the cochlear partition. Thus, we observed greater amplitude of CMWs in click, low tonal, and high tonal stimuli, in descending order. Therefore, click instead of tonal stimuli results in lesser time and larger amplitude, and it is better to measure cochlear evoked potentials with click stimulation as a test approach in some special conditions such as ANSD.